GRP94, an ER chaperone of the HSP90 family, is essential because it controls the folding of secreted and membrane proteins. One unique feature of this chaperone, which makes it a good drug target, is its selectivity: GRP94 controls the expression of only few substrate proteins. Nonetheless, these substrates play critical roles in a number of cell-cell interactions. One such substrate is IGF-I, a mitogenic hormone whose signaling determines growth and differentiation outcomes in many tissues. It also signals in autocrine fashion growth/death decisions in many tumor cells. We discovered that processing and secretion of IGF-I are inhibited in GRP94-deficient cells, showing strict dependence on the activity of GRP94. We took advantage of this finding and developed an assay in which cell viability is conditional on IGF-I secretion and therefore on the availability of active GRP94. This is the first mammalian cell-based assay for the function of GRP94, and indeed any ER chaperone. It will help define distinct functional domains and active sites of GRP94, as well as its interacting proteins. Because of its potential, we propose to develop a high throughput format for this assay, in order to make it suitable for screening small molecule libraries. The goal will be to find probes that inhibit GRP94 selectively, without affecting the homologous protein HSP90, rather than the pan-HSP90 probes that are currently available. To complement this cell-based assay, we will convert to a high-throughput format three protein-based assays that measure distinct activities of GRP94 - peptide binding that is mediated by the N-terminal domain, calcium binding that is mediated by the second, acidic domain, and conversion from an active to inactive conformation, which reports on the dynamics of both the N-terminal and middle domains. This will allow us to relate the activity of any probe in the cell-based assay to the in vitro activities, and thereby we will not only verify the specificity of the probe, but also gain knowledge of its mechanism of action. Like any chaperone, the action cycle of GRP94 involves dynamic intra- and inter-molecular interactions mediated by multiple domains. A high throughput screen of molecular libraries using functional assays is the best approach to obtain probes defining these multiple interactions, an important step towards understanding why GRP94 is an essential mammalian protein. GRP94 is an essential molecular chaperone that controls the secretion of specific proteins, including growth factors that are important for normal development as well as for the progression of many tumors. The overall goal of this proposal is to develop assays can be used to screen small molecule libraries to discover GRP94 specific drugs. If successful, these studies will be valuable for the development of novel therapeutic approaches based on modulating GRP94 function. [unreadable]